PURPOSE: The purpose of this study was to evaluate the influence of methods of establishing the midsagittal reference plane (MRP) on the locations of midfacial landmarks in the three-dimensional computed tomography (CT) analysis of facial asymmetry. MATERIALS AND METHODS: A total of 24 patients (12 male and 12 female; mean age, 22.5 years; age range, 18.2-29.7 years) with facial asymmetry were included in this study. The MRP was established using two different methods on each patient's CT image. The x-coordinates of four midfacial landmarks (the menton, nasion, upper incisor, and lower incisor) were obtained by measuring the distance and direction of the landmarks from the MRP, and the two methods were compared statistically. The direction of deviation and the severity of asymmetry found using each method were also compared. RESULTS: The x-coordinates of the four anatomic landmarks all showed a statistically significant difference between the two methods of establishing the MRP. For the nasion and lower incisor, six patients (25.0%) showed a change in the direction of deviation. The severity of asymmetry also changed in 16 patients (66.7%). CONCLUSION: The results of this study suggest that the locations of midfacial landmarks change significantly according to the method used to establish the MRP.